Intraocular delivery of bioactive molecules using iontophoresis

ABSTRACT

Iontophoresis, a minimally-invasive methodology that uses a weak electric current to enhance penetration of ionized molecules into tissues, was found to be an effective technique for the intraocular delivery of large bioactive molecules, specifically lutein.

This application is a continuation of U.S. patent application Ser. No.14/977,048, filed Dec. 21, 2015, and entitled INTRAOCULAR DELIVERY OFBIOACTIVE MOLECULES USING IONTOPHORESIS, which claims priority to U.S.Patent Application Ser. No. 62/094,663, filed Dec. 19, 2014, entitledINTRAOCULAR DELIVERY OF BIOACTIVE MOLECULES USING IONTOPHORESIS, both ofwhich are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

The present invention relates generally to the use of bioactivemolecules to ocular tissues and, more specifically, to the delivery oflutein to the macula using iontophoresis.

Lutein (and zeaxanthin) are associated with reducing the risk ofdeveloping AMD (Age-related Macular Degeneration) and cataractextraction due to its antioxidant and photoprotective effects, and itsexclusive distribution in the eye macula¹. Age is one of the mostimportant risk factors for AMD, typically affecting individuals over 50years old²⁻⁴. There are two types of AMD, ‘dry AMD’ and ‘wet AMD’. DryAMD develops when macular cells become damaged as a result of wasteproduct accumulation called “drusen”. It is the most common and leastserious type of AMD. An estimated high number of those that present dryAMD symptoms will develop wet AMD, which develops when abnormal bloodvessels from underneath the macula grow and lead to irreversible celldamage²⁻⁴.

Lutein has been widely used through oral supplementation with therationale that systemic circulation can bring lutein to the coroidalcirculation for uptake into the macula, through xanthophyll-bindingproteins⁵. However, several reports demonstrate that only a smallpercentage of lutein reaches the macula⁶⁻⁸. Moreover, due to eye barrierlimits, therapeutic treatments in the posterior eye segment aredifficult. Since the eye is protected by the tear film, corneal,vitreous, blood-retinal and blood-aqueous barriers, it is very difficultto deliver drugs to the eye, particularly to the retina, in sufficientconcentrations and with minimal side-effects^(9,10). In-situapplications have been used to overcome this problem; however, slowdelivery systems, such as implants, are very invasive and expensive.Over the past few years, the results of many studies have highlightedthe risks of these treatments^(11,12).

Recently, intra-vitreous injections of lutein have been used to stainspecific preretinal membranes and other eye structures duringsurgery¹³⁻¹⁶. This has been the first data on in-situ delivery of luteintowards the macula, exploiting lutein's intrinsic staining effect.Lutein's potential of delaying AMD progression and putativeneuroprotective action shown in different trials has not yet been proventhrough in-situ application following intraocular delivery.Intravitreous injection of lutein for a prevention purpose may be tooinvasive as a strategy of delivering lutein to the macula, with thedisadvantage of poor patient acceptance.

Ocular iontophoresis is a minimally invasive method used to propel byelectrical force high concentrations of target molecules transsclerallyor/and transcorneally. It uses a small electrical current applied to aniontophoretic chamber containing the molecule of interest and vehicle¹⁷.Several reports revealed that lutein, which is found in highconcentrations in the macula of the human eye, has the potential ofdelaying AMD progression, in addition to potential neuroprotectiveaction¹⁸⁻²⁰.

Here we report a novel way of delivering lutein to the retina, so itspresence in the parafovea macular region can be enhanced significantlyand thereby delay the progression of AMD and protect retinal endothelialcells. Different iontophoresis delivery systems for ophthalmic use havebeen created and have been used to safely and effectively delivermedication to both the anterior and posterior segments of the humaneye²¹. With this technology, it is possible to deliver significantamounts of bioactive molecules, including macromolecules, across thecornea and sclera. In the work reported here, a lutein emulsion has beendiffusively delivered to the macula by iontophoresis²²⁻²³. The idea wasto develop a minimally invasive method of propelling high concentrationsof charged lutein, transclerally or/and transcorneally by iontophoresis.We have assessed the distribution and concentration of lutein in thedifferent ocular tissues using two-photon microscopy, Raman spectroscopyand HPLC after scleral and corneal iontophoretic application. The mainadvantage of this approach is to use of a medical device that is saferand easier to have patient compliance, avoiding the complications offrequent and high dose injections or surgical implantations. Thisprocedure can be performed quickly in the doctor's office during anormal eye care appointment with no need of a surgical environment.

SUMMARY OF THE INVENTION

Iontophoresis, a minimally invasive methodology that uses a low electriccurrent to enhance penetration of ionized compounds into tissues, wasfound to be effective for the intraocular delivery of lutein. Fourteenpigmented rabbits were treated by application onto the cornea and scleraof an iontophoretic reservoir filled with lutein emulsion with orwithout current (20.0 and 0.0 mA, respectively). After iontophoresis,the ocular tissues from both eyes (test and control) were collected andlutein delivery was assessed by visual comparison between treated eyeand untreated contralateral eye. The transcorneal and transscleraliontophoresis application resulted in the delivery of lutein to therabbit cornea in all treated eyes (time 0 h). The application of luteinalso created an orange trace on the sclera limbus and a slight orangecoloration in the eye conjunctiva, demonstrating the transport of theemulsion also to these tissues. In this work we have shown for the firsttime that iontophoresis is an effective technique for intraoculardelivery of lutein.

In the present invention, lutein distribution in the eye afteriontophoresis procedure was assessed to confirm that high quantities oflutein were delivered to the posterior retina by this technique. Resultsindicate that iontophoresis is an effective method of delivering apositively charged liposomal emulsion of lutein into rabbits eyes.Furthermore, experiments were performed using optimized formulations oflutein emulsion and an alternative iontophoretic prototype to evaluatelutein distribution in different eye tissues.

Trials were also performed with human cadaveric eyes to which a lowelectric current was applied to evaluate lutein delivery through thecornea and sclera. The cornea, sclera, choroid, peripheral and centralretina from treated and non-treated eyes were collected and analyzed bytwo-photon microscopy in order to visualize the distribution oflutein-containing liposomes.

The transscleral iontophoretic application resulted in the delivery ofthe lutein mainly to the posterior retina region, revealing the pathwayof lutein after the iontophoresis occurs via ciliary body/pars planafollowed by passive diffusion until reaching the posterior retina. Theabsence of lutein in the choroid can be explained by the narrow size oftight junctions of the retinal pigmented epithelium, which impair thepassage of the larger liposomal vesicles, thereby trapping lutein in theretina inner layers.

With this work we demonstrated for the first time the in situ deliveryof lutein to the posterior eye segment through a novel, minimallyinvasive method. The results demonstrate that scleral iontophoresis oflutein is an effective strategy of delivering lutein to the macula,which represents an alternative to the current methods used to delaydiseases in the posterior eye segment, such as AMD.

Iontophoresis has the advantage of being a minimally invasive methodand, therefore, is safer than the alternative methods of intraoculardelivery of compounds, namely implants and intra-ocular injections.Consequently, iontophoresis will have a higher patient compliance sinceit avoids the complications of a surgical implantation or frequent andhigh dose intravitreal injections. Another advantage is this techniqueis less expensive than those procedures and can be performed quickly inthe doctor's office during a normal eye care appointment with no needfor a surgery environment.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a chart of Lipo+ absorption spectra (300 to 750nm), 1:50dilution in 0.9% NaCl (blue) or distilled water (red); the controlspectrum (liposome solution without lutein) is represented.

FIG. 2 is a chart of Lipo+ fluorescence spectra (480 to 650nm), 1:50dilution in 0.9% NaCl (blue) or distilled water (red); the controlspectrum (liposome solution without lutein) is represented.

FIG. 3 is a chart of the dynamic light scattering and electrophoreticmobility to estimate particle size distribution and charge of Lipo+solution: a 1:50 dilution in distilled water (red) was tested and also1:50 liposome dilution in water (without lutein) as control.

FIG. 4 is a schematic representation of lutein trajectory afteriontophoresis application; scleral application deposits lutein to theback of the eye, whereas corneal application leaves lutein sitting ontop of the corneal epithelial cells (orange spots); arrows indicate theentrance of lutein following iontophoresis application.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The terms “administration of” or “administering a” compound should beunderstood to mean providing a compound of the invention to theindividual in need of treatment in a form that can be introduced intothat individual's body in a therapeutically useful form andtherapeutically effective amount, including, but not limited to: oraldosage forms, such as tablets, capsules, syrups, suspensions, and thelike; injectable dosage forms, such as IV, IM, or IP, and the like;transdermal dosage forms, including creams, jellies, powders, orpatches; buccal dosage forms; inhalation powders, sprays, suspensions,and the like; and rectal suppositories.

The term “effective amount” as used herein refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof a composite or bioactive agent may vary depending on such factors asthe desired biological endpoint, the bioactive agent to be delivered,the composition of the encapsulating matrix, the target tissue, etc.

As used herein, the term “extract” refers to a product prepared byextraction. The extract may be in the form of a solution in a solvent,or the extract may be a concentrate or essence which is free of, orsubstantially free of solvent. The term extract may be a single extractobtained from a particular extraction step or series of extraction stepsor the extract also may be a combination of extracts obtained fromseparate extraction steps. For example, extract “a” may be obtained byextracting cranberry with alcohol in water, while extract “b” may beobtained by super critical carbon dioxide extraction of cranberry.Extracts a and b may then be combined to form extract “c”. Such combinedextracts are thus also encompassed by the term “extract”.

As used herein, the term “fraction” means the extract comprising aspecific group of chemical compounds characterized by certain physical,chemical properties or physical or chemical properties.

The term “preventing”, when used in relation to a condition, such ascancer, an infectious disease, or other medical disease or condition, iswell understood in the art, and includes administration of a compositionwhich reduces the frequency of, or delays the onset of, symptoms of amedical condition in a subject relative to a subject which does notreceive the composition. Thus, prevention of cancer includes, forexample, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population.

By “pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The term “synergistic” is well understood in the art and refers to twoor more components working together so that the total effect is greaterthan the sum of the components.

The term “treating” is well understood in the art and refers to curingas well as ameliorating at least one symptom of any condition ordisorder.

The term “prophylactic or therapeutic” treatment is well understood inthe art and includes administration to the host of one or more of thesubject compositions. If it is administered prior to clinicalmanifestation of the unwanted condition (e.g., disease or other unwantedstate of the host animal) then the treatment is prophylactic, i.e., itprotects the host against developing the unwanted condition, whereas ifit is administered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The compounds of this invention may be administered to subjects (humansand animals, including companion animals, such as dogs, cats and horses)in need of such treatment in dosages that will provide optimalpharmaceutical efficacy. It will be appreciated that the dose requiredfor use in any particular application will vary from patient to patient,not only with the particular compound or composition selected, but alsowith the route of administration, the nature of the condition beingtreated, the age and condition of the patient, concurrent medication orspecial diets then being followed by the patient, and other factorswhich those skilled in the art will recognize, with the appropriatedosage ultimately being at the discretion of the attendant physician.

EXAMPLE 1 Intra-ocular Delivery of Lutein in Rabbit Eyes Materials andMethods

Formulation work. Among the different delivery systems currently used toimprove the stability of compounds, liposomes have advantages due totheir biocompatibility, sustained release potential, and the ability tocarry both hydrophobic and hydrophilic compounds¹⁶. In this work,crystalline lutein (Kemin Foods, FloraGLO® Crystalline Lutein lot.1401103302) was encapsulated in liposomes using phospholipids 90H(Lipoid GmbH, lot 529400-2120046-12-112, CAS 308068-11-3) andoctadecylamine (Sigma-Aldrich lot BCBK6340V, CAS 124-30-1). Lipid filmwas prepared using 90H phospholipids, octadecylamine and luteindissolved in CHCl₃/MeOH (2:1) (Sigma-Aldrich, lot SHBC4982V, CAS67-66-3/Sigma-Aldrich, lot SZBC237BV, CAS 67-56-1). Solvents wereremoved under vacuum by rotary evaporation; the solution was dried undervacuum at 40° C. by a Heidolph rotavapor, the speed of the rotavapor wasmodulated in order to reduce bubble formation and splashing that couldcause loss of product and a dry thin film was obtained after 1-2 hours.To remove any trace of solvents, the thin film was left under vacuum forat least 16 hours at room temperature. Lipid film hydration wasperformed by adding distilled water (Water Ultrapure—MilliQ-byAquaMax—conductivity 0.054 uS/cm) at 40-45° C. to the lipid film tohydrate lipids and form large liposome vesicles. The homogenization ofthe large liposome vesicles was achieved using Ika Works ULTRA-TURRAX T25 Digital Homogenizer (Staufen, Germany), and reduction of liposomevesicles to a nano size range has been performed by extrusion usinglarge-scale Microfluidizer® high fluid processor M-110EH at 50-60° C.and 1200 bar. This process was repeated 5 times. Sterilization of theemulsion was performed at 121° C. for 20 minutes at 1 atm. Table 1 showsliposome emulsion composition. Size distribution, zeta potential,osmolality and pH of the final product were analyzed after sterilizationand are summarized in Table 2.

TABLE 1 Lutein liposome emulsion composition. Composition % w/w 90Hphospholipids 1.000 Octadecylamine 0.005 Lutein crystals 0.050 Distilledwater to 100 g

TABLE 2 Liposome characteristics after sterilization. Osmolality Meandiameter Zeta potential pH (mOsM/kg) (nm) (mV) 6.84 15 194 +36.93

Ocular iontophoresis device. The iontophoresis device consisted of twodisposable components: an ocular applicator and a return electrode.These two components were connected to a reusable generator. The ocularapplicator was composed by a polycarbonate reservoir (diameter 9 mm,height 4.5 mm, volume 0.5 ml) and a stainless steel electrode (AISI 304)connected with a lead to the generator (anode—positive electrode). Thereturn electrode was a 25G intradermic needle, inserted in the neck(front side) and connected with a crocodile clip and lead to thegenerator (cathode). The generator (EYEGATE CCI Generator 6121-EYE,Eyegate Pharma, Paris France) was a constant current type, setting range0.25 mA-2.5 mA (10 increments of 0.25 mA) for the current and 0.5 min-5min for the time (10 increments of 0.5 min). The resulting voltageapplied was measured during the study with a multimeter.

Animals. Fourteen pigmented rabbits strain HY79b (Breeder:“HYPHARM”—FR-49450 ROUSSAY) were used in this study. All animals wereidentified individually using an ear tag and using a marker in the earsfollowing the inclusion examination. Animals were held in observationfor 3 days following their arrival, and were daily observed for signs ofillness with particular attention to the eyes. Animals were individuallyhoused in standard cages, under identical environmental conditions. Thetemperature was held at 15-21° C. and the relative humidity at 55±10%.Rooms were continuously ventilated (≥15 air volumes per hour).Temperature and relative humidity were continuously controlled andrecorded. Animals were routinely exposed (in-cage) to a 10-200 l× lightin a 12-hour light (from 7:00 a.m. to 7:00 p.m.) and 12-hour darknesscontrolled cycle. Throughout the study, animals had free access to foodand water. They were fed a standard dry pellet diet (150 g/day),LASQCdiet® Rab-14H (LASVENDI GMBH, Soest Germany). Tap water, regularlyanalysed, was available ad libitum from plastic bottles. All standardoperating procedures and protocols described in this study plan havebeen reviewed by a certified Ethical Committee. All animals were treatedaccording to the Directive 2010/63/UE European Convention for theProtection of Vertebrate Animals used for Experimental¹⁷ and OtherScientific Purposes and to the Association for Research in Vision andOphthalmology (ARVO) Statement for the Use of Animals in Ophthalmic andVision Research¹⁸.

Experimental procedure. Fourteen pigmented rabbits from HY79b strainwere randomly divided into two groups: control (passive application:without electric current; animals #9-14) and test group (iontophorecticapplication: with electric current; animals #1-8). These two groups weresubdivided in two time-points (0 and 2 hours). Table 3 summarizes thestudy design.

TABLE 3 Study design. Group No. Drug Administration Time-points Animalsid# 1 Lutein Iontophoretic delivery 0 h 1, 2, 3, 4 2 emulsion (charge =20.0 mA) 2 h 5, 6, 7, 8 3 Iontophoretic delivery 0 h 9, 10, 11 4 (charge= 0.0 mA) 2 h 12, 13, 14

Lutein emulsion was administered by iontophoresis to anesthetizedanimals (intra muscular injection of a mix xylazine/ketamine), aidedwith a blepharostat and under local anesthesia (one drop of Cebesine®:0.4% oxybuprocaïne, Thea, lot F6757) about 10 min before application).Animals were treated by application onto the cornea and sclera of a 9-mmiontophoretic applicator filled with lutein for 10 minutes on right eye.A charge of 0.0 mA or 20.0 mA was applied on each eye, depending on thegroup (see Table 3). The iontophoretic applicator was impregnated with0.5 mL of lutein liposome emulsion just before dosing; the electrode ofthe device was covered with lutein emulsion. All administrations werefollowed by balanced salt solution (BSS) washing.

Immediately after the iontophoretic application of the right eye or 2hours post-application (see Table 3), animals were euthanized byintravenous administration of overdosed pentobarbital, which is amongthe recommended methods by the European Authorities¹⁷. Cornea (C),aqueous humor (AH), ciliary-body (CB), retina (R), vitreous (V) andsclera (SC) from both eyes were sampled and weighed. A visual evaluationof the coloration of the samples was performed before storing them at−80° C. for future HPLC (high-performance liquid chromatography)analysis.

Results

Ocular iontophoretic delivery of lutein emulsion in pigmented rabbits.In order to evaluate the capacity of lutein to be delivered byiontophoresis, we produced liposomes carrying lutein (positivelycharged) and applied this emulsion for 10 min with 2.0 mA into thecornea/sclera of pigmented rabbits. The efficacy of delivery byiontophoresis was evaluated by visual assessment of the collectedtissues. Table 4 summarizes the results after the application of luteinemulsion, with and without current.

TABLE 4 Coloration after iontophoretic application. Tissue Ocular tissuecoloration (upon sampling) Rabbit collection Untreated eye Treated eyeID# Iontophoresis time-point (left) (right) 1 2.0 mA charge 0 h Nocoloration Cornea: slight circular orange for 10 min trace 2 Nocoloration Cornea: circular orange trace. SC: orange trace on thelimbus. CJ: slight orange coloration 3 No coloration Cornea: circularorange trace. SC: orange trace on the limbus. CJ: slight orangecoloration 4 No coloration Cornea: circular orange trace 5 2 h Nocoloration Cornea: circular orange trace 6 No coloration No coloration 7No coloration No coloration 8 No coloration Cornea: circular orangetrace 9 10 min 0 h No coloration No coloration 10 application Nocoloration No coloration 11 without charge No coloration No coloration12 2 h No coloration No coloration 13 No coloration No coloration 14 Nocoloration No coloration Note: C = Cornea; SC = Sclera; CJ = Conjunctiva

Subsequently to the iontophoresis application, all the eyes treated with20.0 mA of current (time 0 h) revealed a circular orange color in thecornea revealing the present of lutein emulsion in the tissues. Theapplication of lutein also originated in two eyes (#2 and #3) an orangetrace on the sclera limbus and a slight orange coloration in the eyeconjunctiva. After 2 h of treatment only half of the treated eyes showedthis coloration in the cornea (#5 and #8), this event may indicate thatsubsequently to the application, the emulsion diffuses into the eye. Nodelivery into the different ocular tissues was observed without current.

Discussion

Approximately 10% of people over 65 years around the world suffer fromAMD disease° . Different trials have indicated lutein is a potential AMDprogression delayer and also a potential neuroprotectivemolecule^(13, 20-22). Moreover, lutein is a natural component of theeye, with intrinsic macular tropism, being specifically deposited in thepara-foveal area where it is congenital¹. These features can be anadvantage towards the current products used to control AMD. Theavailable treatments for this pathology involve intraocular injectionsthat have side effects, are troublesome to the patient and expensive, sothe development of a more safe and effective treatment is crucial. Overthe past few years, results of many studies have highlighted the risksof intravitreal injections. The need for frequent administration ofdrugs through intravitreal injections can lead to retinal detachment,endophthalmitis and increased intraocular pressure. Both noninfectiousand infectious inflammation has been reported as complications ofintravitreal injections. With the increasing rates of intravitrealinjections since their approval for use, the incidence of infectiousendophthalmitis has been extensively studied^(23,24). In this work wetested, for the first time a minimally invasive technology to deliverlutein in-situ. Iontophoresis has the advantage of being a minimallyinvasive method and therefore is safer and easier to improve patientcompliance, since it avoids the complications of a surgical implantationor frequent and high dose of intravitreal injections¹². In factdifferent pre-clinical and clinical studies reported the safety ofrepeated ocular iontophoresis applications^(14, 25, 26). Anotheradvantage is this method is less expensive and can be performed quicklyin the doctor's office during a normal eye care appointment with no needfor a surgery environment. Different studies established the use ofiontophoresis for the treatment of human eye diseases, for instance inmanagement of active corneal graft rejection²⁷, treatment of dry eyedisease^(14, 28), noninfectious anterior uveitis¹⁵ and keratoconusdisease²⁹.

In this investigation we have used iontophoresis that involves theapplication of a weak direct current during 10 minutes that drivescharged molecules across the eye tissues. The iontophoretic applicationresulted in the penetration of the emulsion of liposomes carrying lutein(ionized drug) through the corneal segment of the eye.

This study is an effective proof-of-concept that clearly shows anintraocular delivery of lutein emulsion through iontophoresis technique.

EXAMPLE 2 Intra-ocular Delivery of Lutein in Cadaveric Eyes Materialsand Methods

Formulation work. It has been demonstrated that positive particles arebetter candidates for iontophoretic application as drug carrier than thenegatively charged particles due to higher penetration into oculartissues³⁸. Furthermore, the electrical field forces the positive chargedmolecules to move into eye membranes (negatively charged)³⁹. In thiswork we took advantage of the fact that the membranes present in thehuman eye, at physiological pH, are negatively charged and fordeveloping a positively charged emulsion carrying lutein to be deliveredthrough iontophoresis application. Due to the fact that lutein is amolecule with a large molecular weight, lipophilic and insoluble inwater, the delivery of this carotenoid trough iontophoresis withoutmodifications is nearly impossible¹. In order to overcome that, aformulation with positively charged liposome vesicles that behave ascarriers of lutein molecules was prepared (Lipo+).The lipid film wasprepared using phospholipon 90H (Lipoid GmbH, lot 529400-2120046-12-112,CAS 308068-11-3), octadecylamine (Sigma-Aldrich lot BCBK6340V, CAS124-30-1) crystalline lutein (Kemin Health, FloraGLO® Crystalline Luteinlot. 1401103302). For preparation of 4-5 L, the compounds were dissolvedin 500-800 mL of CHCl₃/MeOH (1:1 v/v) (Sigma-Aldrich, lot SHBC4982V, CAS67-66-3/Sigma-Aldrich, lot SZBC237BV, CAS 67-56-1) by heating at 30-35°C. Please see formulation composition in Table 5. Solvents were removedunder vacuum by rotary evaporation; the solution was dried under vacuumat 40° C. by a Heidolph rotavapor, and a dry thin film was obtainedafter 1-2 hours. The thin film was left under vacuum for at least 16hours at room temperature to ensure the complete removal of any trace ofsolvents. The content of organic solvents was analyzed by gaschromatography (GC) and was assured to be less than 25 ppm. Lipid filmhydration was performed by adding distilled water (WaterUltrapure—MilliQ-by AquaMax—conductivity 0.054 uS/cm) at 65° C. to thelipid film to form large liposome vesicles. Homogenization of theselarge liposomes vesicles was achieved using Ika Works ULTRA-TURRAX T 25Digital homogenizer (Staufen, Germany) at 2000-4000 rpm and reduction ofliposome vesicles to nano size range has been performed by extrusionusing large-scale Microfluidizer® high fluid processor M-110EH at 50-60°C. and 1200 bar. This process was repeated 5 times. Sterilization of theemulsion was performed at 121° C. for 20 minutes at 1 atm. After thesterilization process, the characteristics of the liposomal formulationwere recorded: pH (using a Mettler Toledo S20 instrument), osmolality(using Osmomat 3000), particle size and zeta potential (using dynamiclight scattering (DLS), also known as photon correlation spectroscopytechnique—Nicomp 380 DLS.

TABLE 5 Lutein liposome emulsion composition. Composition % w/w 90Hphospholipids 2.000 Octadecylamine 0.007 Lutein crystals 0.100 Distilledwater to 100 g

Spectrophotometric evaluation of the formulation. Spectrophotometry wasused to measure the absorbance and fluorescence properties of the Lipo+solution (1:50 dilution in water or 0.9% NaCl). A solution includingonly liposomes (without lutein) was used as control. The absorbancespectra were traced between 300 and 750 nm for each sample and thefluorescence spectra between 480 and 650 nm, with excitation at 370 nm(chosen from the absorbance spectra results).

Particle size and zeta-potential. Dynamic light scattering was used toevaluate the distribution of sizes of the components in the Lipo+solution. Electrophoresis was performed to evaluate the zeta potentialof the solution.

Ocular iontophoretic device. The principle of ocular iontophoresis isapplying an electric field to an electrolytic substance containing atleast one product, in order to transport the product into the body orthe organ to be treated, via the biological membranes of the eye¹².

A typical iontophoretic setting is made of two components: ocularapplicator and a return electrode both connected to a generator. In thisexperiment, the ocular applicator comprised 2 electrodes to addressindependently corneal and scleral tissues. The ocular applicator (OPIATechnologies SAS, Paris, France) is made of polyurethane resin andcomprises 2 reservoirs: central circular reservoir (diameter 8 mm,height 4.5 mm, volume 1 ml) and a stainless steel electrode, applied onthe cornea surrounded by an annular reservoir (inner diameter 12.5 mm,outer diameter 18 mm height 4.5 mm, volume 1 ml) and a stainless steelelectrode, applied on the sclera (pars plana region around the limbus).Each stainless steel electrode connected with a lead to a differentconstant current generator (anode—positive electrode). The returnelectrodes were assigned to each generator and respectively attached tothe optic nerve (for the corneal electrode) and the equator region ofthe sclera (for scleral electrode), closing each electrical circuitindependently. The generators (IONO-25, Iacer Srl, Italy) were aconstant current type, setting range 0.25 mA-2.5 mA (5 increments of 0.5mA) for the current and time adjusted automatically to deliver a totaldose of 20 mA.min. The resulting voltage applied was measured during thestudy for each circuit with 2 multimeters.

Cadaveric eyes. Six human cadaveric eye globes, from different healthydonors, were obtained from the Veneto Eye Bank Foundation (VeneziaZelarino, Italy). The human eyes were used in compliance with theguidelines of the Declaration of Helsinki for research involving the useof human tissue and were explanted between 3 and 16 hours after deathand immediately preserved at 4° C. in corneal storage medium enrichedwith 6% dextran. The mean donor age was 63.6±5.9 years. The meanendothelial cell density was 2125±389 cells/mm². Each eye globe,submerged in dextran enriched solution, was shipped to the laboratorywithin 5 days. Four eye globes underwent corneo-scleral iontophoresis todeliver 0.1% lutein ophthalmic solution into the retinal tissue. Two eyeglobes were used as control: iontophoresis was performed withoutpresence of the formulation.

Preparation of the eyes. Each eye globe was gently mounted into aspecially designed holder, facing upward. The scleral and cornealpassive electrodes were applied in the optic nerve and sclera,respectively. The eye was connected to a column manometer by a tube,filled with 0.9% sodium chloride solution, in order to maintain thepressure inside the eye at 15 mmHg during the experiment. The eye globewas first subjected to three cycles of pre-conditioning between 15 and42 mmHg in order to stabilize the ocular tissues and mechanics duringexperiment. This preconditioning ensured to attain a unique referencestate at the beginning of each experiment and to restore the corneal andscleral thickness to physiological levels. After pre-conditioning, thecentral corneal thickness (CCT) was measured, using an ultrasoundcorneal pachymeter (Pachmate, DGH, Exton, USA). In these samples, themean CCT was 558±19 pm.

Impregnation with the solution. The active electrode (cathode), in aplastic bath, was applied to the corneal and scleral surface. Theplastic tube was filled with foam, which was soaked with Lipo+ for 20minutes. After this pre-soaking treatment, the tube was gently appliedto the anterior surface of the eye globe and again filled with 2 mL ofLipo+ solution. The current density was set at 2.5 mA and delivered for5 minutes for both generators connected to the cornea and sclera. Aftercorneo-scleral iontophoresis, the eye globe was maintained, facingupward, in the eye holder with the pressure inside the eye at 15 mmHgfor 80 minutes. This period allowed the lutein, which reached the retinaby trans-scleral iontophoresis, to diffuse passively, through theretinal tissue, towards the macula, specially the para-foveal region.Two of the 6 eyes used in this study were used as control, thus noimpregnation with the formulation was performed.

Tissue evaluation. After 80 minutes, the retinal tissue was isolatedwithout inducing gross damage that could compromise their use forhigh-resolution two-photon imaging. Dissection of retinal, choroidal,corneal and scleral tissues was done using a standardized protocol⁴⁷.Two-photon microscopy was used to evaluate penetration of lutein in themacular region of the retina. Before starting image acquisition onocular tissues, several stacks on Lipo+ solution (0.005%, 0.002% and0.001% dilutions in 0.9% sodium chloride) were acquired in order tounderstand the best filter to apply and enhance the two-photonfluorescence (TPF) signal emitted by lutein. The filter 550/80 nm(Semrock) was the most appropriate for the study of lutein (based inspectrophotometric studies) however, the filter 525/20 nm (Semrock) wasfound to give good results in terms of Signal-to-noise ratio (SNR).Therefore, the excitation used for ocular tissues evaluation was 835 nmand TPF light emitted by ocular tissue components was collected inbackward direction by a non-descanned detector (NDDI) for reflectedlight reflection.

Resonance Raman Spectroscopy: Resonant Raman scattering was used forevaluating the efficacy of iontophoresis delivery of lutein to the humanretina in cadaveric eyes. A single-mode laser source (50 mW power),centered at 473.5 nm wavelength, was used as excitation source toperform resonance Raman spectroscopy measurement. The laser beam wasfocused on the ocular tissues by a combination of lenses and amicroscope objective (NA=0.25), so that the irradiated retina area was 1mm in diameter; the laser power was reduced to 1 mW at the retinal planeusing a neutral density filter. The Raman scattered light was collectedby a photomultiplier tube (PMT), with spectral resolution of 10 cm⁻¹ andwith an average of 80 dark counts rate. Raman signal intensity wasrecorded as photon counts per second (cps). Measurements were performedon three retinal regions: the inner sclera at the site of iontophoreticdelivery (i.e., the perilimbal sclera facing towards the ciliary body);the retinal mid-periphery, which included the region of the retinasurrounding the vascular arcades and the optic nerve head; and themacula. Measurements were performed in four areas across each region inorder to collect enough data to correctly estimate data in the study andcontrol eyes. Before the experiment, in order to find a correlationbetween the Raman readings and the actual lutein content of the oculartissues, calibration experiments using thin quartz cuvettes filled withdifferent concentration of lutein were performed.

Results

Lipo+ spectral characteristics. Absorbance and fluorescence spectra ofLipo+ solution were initially addressed in this study in order todetermine the two-photon excitation wavelength. The absorption spectrawere traced between 300 and 750 nm and are represented in FIG. 1. TheLipo+ solution showed an absorption peak at 370 nm and this was used asexcitation wavelength to trace Lipo+ fluorescence spectra between 480and 650 nm (FIG. 2). Lipo+ showed two fluorescent band peaks: 500-530 nmand 540-570 nm. Based on these results, the filter chosen for thetwo-photon experiments was the 550/88.

Lipo+ physical characteristics. Particle size and zeta-potential werealso determined before iontophoresis testing, in order to confirm Lipo+positive charge and size. These determinations were performed throughdynamic light scattering and electrophoresis, respectively, for a 1:50Lipo+ dilution in distilled water, and were also compared to thelutein-free liposome solution (as control). According to FIG. 3, Lipo+aggregates peak at 3.5 μm (in average) and a smaller peak is also seenat 300 nm (in average), indicative of the individual liposomes. Also,the zeta potential determination showed a +5 mV charge for the Lipo+solution.

Lutein distribution in cadaveric eyes after iontophoretic application.Although the filter chosen for the two-photon experiments was the 550/88(based on previous fluorescence experiments), initial analysis of thelutein liposomal formulation revealed that the 525/20 filter gave betterresults in terms of SNR. In this initial calibration (with 0.005, 0.002and 0.001% Lipo+ dilutions in 0.9% % NaCl), liposomes were observed asspherical vesicles and the microscope was calibrated correctly (data notshown). This is a very important control because the retinal pigmentcells are full of melanin, a pigment that excites at the same wavelengthas lutein. Moreover, with this control we are able to distinguishbetween the lutein liposomes and the pigment.

In order to assess the distribution of the liposomes carrying lutein inhuman eye after iontophoretic application, five cadaveric eyes wereexposed to a current of 2.5 mA for 5 min into the cornea/sclera, allowedto rest for 80 min (controlled intraocular pressure at 15 mmHg) and thedifferent structures of the eye were collected: cornea, sclera, choroid,peripheral and central retina. The distribution of the liposomes wasevaluated by two-photon microscopy (excitation at 835 nm). A sixthcadaveric eye was used as control: the eye was never in contact with theliposomal formulation. In this, no liposomes were detected when the 835nm laser was on, indicating that the signal is specific to exogenouslutein (data not shown).

Analysis of the different eye tissues collected showed that aftercombined corneo-scleral iontophoresis, lutein was abundant in theretina, while no lutein-enriched liposomes were found in the choroidaltissues for all tested eyes with the formulation.

Also, from the retinal investigation, Lipo+ solution was not able tocross the wall of retinal vessels, since liposomes were only found inthe tissue surrounding the vessels.

For the anterior segment determinations (sclera and cornea), nolutein-enriched liposomes were found, neither in the corneal stroma norin the sclera tissue, but were found in corneal epithelial cells

In the retina it was also possible to observe more lutein in the outerpart close to the photoreceptors than in the ganglion cells, the innerpart of the retina.

Since the choroid analysis revealed no liposomes were present in thisregion after the current application, these results indicate that aftertransscleral application of lutein by iontophoresis via ciliarybody/pars plana, lutein liposomes diffuse passively through the eyemembranes until reaching the posterior retina near the fovea. FIG. 4shows a schematic representation of the lutein pathway within the eye.Analysis of corneal tissue following corneal iontophoresis application,revealed the liposomes were sitting on top of the epithelial cells ofthis tissue.

Resonance Raman spectroscopy analysis of different eye tissues was alsoperformed. Raman signals were superimposed on a fluorescence backgroundlikely originating from intrinsic carotenoid fluorescein and lipofuscinfluorescence. To obtain an accurate reading of the Raman peak heights,free of background signals, we subtracted the influence of potentiallyoverlapping noise spikes in the spectrum by polynomial fitting (up to5th order) of the measured Raman line shapes for each measured spectrum.The final peak height of the C═C double bond signal at 1530 cm-1 waschosen as a signature of the presence of lutein.

In the inner sclera, the Raman peak at 1530 cm-1 measured in a treatedeye was 7 times greater than control eye, providing the evidence ofefficacy of iontophoresis in delivering lutein to the eye through theintact sclera. In the retinal mid-periphery, the Raman peak at 1530 cm-1measured in a treated eye was 1.7 times greater than control eye, whichindicated that a large amount of lutein reached the posterior pole ofthe retina at the end of iontophoresis treatment. In the macula, theRaman peak at 1530 cm-1 measured in a treated eye was 1.3 times greaterthan in controls, demonstrating that iontophoresis was effective indelivering lutein in the macula.

Discussion

Age-related macular degeneration (AMD) is the leading cause ofirreversible blindness in people over 50 years in the developedworld^(40, 41). More than 8 million Americans have AMD, and the overallprevalence of the disease is projected to increase by more than 50% bythe year 2020³⁷. Several epidemiological studies highlighted that luteinsupplementation lead to an increase in the macular pigment opticaldensity (MPOD) levels in early-stage AMD patients, being associated withprotection from macular disease^(42, 21). In fact, lutein is naturallyconcentrated in the retina, where together with zeaxanthin forms themacular pigment. Acting as a blue light filter, lutein can protect theunderlying photoreceptors in the center of the macula from photochemicaldamage⁴³. The anti-oxidant properties of lutein may also protect themacula from oxidative stress⁴⁴.

The available solutions to slow AMD progression are based on intraocularinjections or surgeries, encompassing evidenced side effects andpossible complications such as retinal detachment, increased intraocularpressure and also noninfectious and infectious inflammation^(23,33). Inthis work, we used a minimally invasive in situ delivery of lutein tothe posterior segment of the human eye. Iontophoresis has the advantageto be safer and easier method to have patient compliance and propellinghigh concentrations of a product of interest through the different eyelayers until it reaches the retina. Different reports have establishedthe safety of repeated treatments using ocular iontophoresis for thetreatment of different diseases such as dry eye, noninfectious uveitisand keratoconus14,15,25,26,28,29.

Herein, using cadaveric eyes as pre-clinical model we applied a weakelectric current to propel lutein into the eye, without side effects. Weobserved that lutein liposomes are mainly deposited in the peripheraland central retina near the fovea, but were absent from the choroidalregions. With this observation it is possible to extrapolate the pathwayof lutein after a transscleral application is via ciliary body/parsplana, followed by passive diffusion through the ocular membranes untilreaching the posterior retina region (FIG. 4). We proved for the firsttime that transscleral iontophoresis is an effective way of bringinglutein to the retina of the human eye providing a new way of fortifyingthe macular pigment. Upon deposition in the posterior region, it ispostulated that lutein is able to reach the outer part of the retinawhere the photoreceptors are present, by passive diffusion/proteingradient. It can be argued the reason why lutein was not observed in thechoroid is due to the neural retinal barrier which mesh size is 80-90nm⁴⁵ (liposomes are 341 nm in size and can sometime form clusters of 2-3μm, suggesting that lutein liposomes stay trapped in the retina (FIG.4). Importantly, resonance Raman spectroscopy analysis revealed thatlutein concentration is increased in the macula after iontophoresis.This observation clearly demonstrates that transscleral iontophoresis isan efficacious method of lutein delivery to the macula and is a validalternative to the current methods for preventing the onset of AMD,prevent its progression and/or treat established disease.

After corneal iontophoresis application no liposomes were present in thecorneal stroma. This fact can be explained since lutein is hydrophobicand the stroma is 70% composed of water, so very hydrophilic⁴⁶, whichmakes impossible for lutein to penetrate in this tissue.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art that have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

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We claim:
 1. A method of depositing a bioactive molecule in oculartissues, comprising the steps of formulating a liposome containing thebioactive molecule, charging an iontophoresis device with a compositionof the bioactive molecule-containing liposome, applying theiontophoresis device to the eye of a subject, and operating theiontophoresis device.
 2. The method of claim 1, wherein the bioactivemolecule is selected from the group consisting of carotenoids.
 3. Themethod of claim 2, wherein the carotenoid is selected from the groupconsisting of lutein and zeaxanthin.
 4. The method of claim 1, whereinthe liposome has a positive zeta potential.
 5. A method of treating,ameliorating or preventing an ocular disease or dysfunction, comprisingthe steps of formulating a liposome containing the bioactive molecule,charging an iontophoresis device with a composition of the bioactivemolecule-containing liposome, applying the iontophoresis device to theeye of a subject, and operating the iontophoresis device.
 6. The methodof claim 5, wherein treating, ameliorating or preventing an oculardisease or dysfunction is delaying the onset of age-related maculardegeneration.
 7. The method of claim 5, wherein treating, amelioratingor preventing ocular disease or dysfunction is preventing or delayingthe progression of age-related macular degeneration.
 8. The method ofclaim 5, wherein the ocular disease or dysfunction is age-relatedmacular degeneration.
 9. The method of claim 5, wherein the amelioratingor preventing of an ocular disease or dysfunction comprises carrying outthe method in healthy subjects.